Ratiometric and Fluorescence-Lifetime-Based Biosensors Incorporating Cytochrome c  ‘ and the Detection of Extra- and Intracellular Macrophage Nitric Oxide

• Published in: Analytical chemistry (Washington) Vol. 71; no. 9; pp. 1767 - 1772
• Main Authors: Barker, Susan L. R, Clark, Heather A, Swallen, Stephen F, Kopelman, Raoul, Tsang, Albert W, Swanson, Joel A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-05-1999
• Subjects: Animals; Arginine - analogs & derivatives; Arginine - pharmacology; Biological and medical sciences; Biosensing Techniques; Biosensors; Biotechnology; Cellular biology; Chemistry; Cytochrome c Group - chemistry; Cytochrome c Group - metabolism; Cytosol - metabolism; Enzyme Inhibitors - pharmacology; Extracellular Matrix - metabolism; Fluorescence; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Hydrogen Peroxide - pharmacology; Hydrogen-Ion Concentration; Interferon-gamma - pharmacology; Lipopolysaccharides - pharmacology; Macrophages - drug effects; Macrophages - metabolism; Methods. Procedures. Technologies; Mice; Mice, Inbred BALB C; Microspheres; Nitric Oxide - analysis; Nitric Oxide - metabolism; Sensors; Spectrometry, Fluorescence - methods; Superoxides - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Various methods and equipments; Cell culture; Fluorescence; Optical sensor; Extracellular; Cytochrome c; Lifetime; Fluorescent labelling; Nitric oxide; Biosensor; Intracellular; Quantitative analysis; Optical fiber; Macrophage
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9810462

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c  ‘, a hemoprotein known to bind nitric oxide selectively. The cytochrome c  ‘ is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c  ‘ attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (≤0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 μM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c  ‘ in solution and have a limit of detection of 30 μM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 ± 90 μM for activated macrophages, while an average intracellular concentration of 160 ± 10 μM was determined from the lifetime-based measurements of dye-labeled cytochrome c  ‘ in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.